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Lipofuscinogenesis in mice early treated with centrophenoxine
Mechanisms of Ageing and Development, 8 (1978) 131-138
131
©Elsevier Sequoia S.A., Lausanne - Printed in the Netherlands
L I P O F U S C I N O G E N E S I S IN M I C E E A R L Y T R E A T E D W I T H C E N T R O PHENOXINE
K. NANDY Geriatric Research, Education and Clinical Center, Veterans Administration Hospital, Bedford, Mass. (U.S.A.)
(Received August 26, 1977;in revised form January 9, 1978)
SUMMARY Previous studies in our and other laboratories indicated that there is a reduction in the neuronal lipofuscin in old rodents after several weeks of treatment with centrophenoxine. The present study investigates whether this chemical can prevent pigment formation if given early in life before the onset of pigmentogenesis. The study shows that the drug did not stop lipofuscin formation in 1 month old mice. But there was a consistent decrease in the pigment in the neurons of cerebral cortex and hippocampus of the treated animals compared to the age-matched controls. The degree of reduction was largely dependent on the duration of the treatment and a significant diminution was noted after treatment for five months or more.
INTRODUCTION Centrophenoxine is synthesized as the hydrochloride of the dimethylaminoethyl ester of p-chlorophenoxyacetic acid. The former component of the drug is a synthetic substance chemically related to the plant growth hormone auxin and the latter is a natural substance in our body and is a precursor of acetylcholine. Clinical trials on geriatric patients in Europe demonstrated a marked improvement of symptoms of senility such as confusion, psychosomatic asthenia and disturbances of memory after treatment for several months with a dose of 80 mg/kg of body weight [1-7]. Its effect on the lipofuscin pigment in the neurons has been demonstrated in the aged guinea pig and mice and a marked reduction of the pigment was noted after treatment for 12 weeks or more [8-18]. In this paper, an attempt has been made to determine whether centrophenoxine can prevent lipofuscin pigment formation completely if the treatment is started in young mice prior to the onset of pigmentogenesis.
132 MATERIALSAND METHODS About 100 old female C57BL/6 mice (1 month) were used in this study. Animals were maintained in constant temperature and humidity controlled environmental chamber and were fed on Purina mouse chow. Fifty female mice (1 month) were treated with centrophenoxine by intraperitoneal injection (80 mg/kg of body weight) daily for 2-11 months and a similar number of the same age and sex and injected intraperitoneally with normal saline were used as controls. Five mice of the control groups were sacrificed at the beginning of the experiment and five mice belonging to each of the control and treated groups were sacrificed after 2, 5, 8, and 11 months of treatment. After the animals were sacrificed, their brains were dissected out and fixed in 10% neutral buffered formalin. Frozen 10/am sections were cut in a cryostat and lipofuscin pigment was visualized by its characteristic autofluorescence by exposing the section to u.v. light at a wavelength of 365 nm. The periodic acid-Schiff and Nile blue sulfate methods also demonstrated the pigment. Semi-quantitative measurements of the pigments in sections were carried out by counting the number of intersections overlying the pigment particles in each of ten sections of frontal cerebral cortex and hippocampus using an ocular grid in a comparison microscope (Duostar, AO).
Fig. 1. Nile blue sulfate stain for lipofuscin in the neurons of the cerebral cortex of a 6 month old control mouse. (x600)
133 RESULTS There were hardly any lipofuscin pigment in the neurons of the cerebral cortex and hippocampus in 1 month old mice. The pigment formation began to appear at 2-3 months of age and thereafter increased progressively as a function of age. The pigment was diffusely distributed in the cytoplasm of the neurons at 2-5 months of age and tended to form perinuclear or polar clumps as the animals grew older. The pigment was primarily visualized by its characteristic autofluorescence and also by histochemical staining with PAS and Nile blue sulfate. In general, lipofuscin was consistently observed in larger quantities in the neurons of the hippocampus than in the cerebral cortex. The treatment with centrophenoxine was started at the age of 1 month when the neurons of cerebral cortex and hippocampus demonstrated very little lipofuscin. Five mice were sacrificed after 2, 5, 8, and 11 months o f treatment and lipofuscin pigment was measured by the semi-quantitative method using an ocular grid in a microscope. The amount of pigment was uniformally lower in all treated groups compared to the controls o f the same age by all the methods employed (Figs. 1--4). While lipofuscin was demonstrated in only 3 out of 5 mice in'the treated group at 3 months of age, all control animals exhibited the pigment in the neurons. It was specially noted that the difference in the pigment content in the treated and control mice was most marked after 11 months of treatment and was statistically significant after treatment for 5 months or more (Table I).
Fig. 2. Nile blue sulfate stain for lipofuscin in the neurons of the cerebral cortex of a 6 month old mouse treated with centrophenoxine for 5 months. (X 600)
134
Fig. 3. Nile blue sulfate stain for lipofuscin in the neurons of the cerebral cortex of a 12 m o n t h old control mouse. (× 600)
TABLE I LIPOFUSCIN PIGMENT COUNTED BY OCULAR GRID METHOD IN THE NEURONS OF C E R E B R A L CORTEX AND HIPPOCAMPUS OF MICE T R E A T E D WITH CENTROPHENOXINE AND AGE-MATCHED CONTROLS
Control group Age (months)
Treated group Neurons of frontal cortex +- 0.34
Neurons of hippocampus
0.6
+- 0.66
Neurons of frontal cortex
Neurons of hippocampus
Duration of treatment (months)
-
-
-
1
0.4
3
4.80 -+ 0.34
6.96 +- 1.08
2.96 -+ 0.98* (P = 0.4)
4.76 ± 1.60" (P = 0.2)
2
6
10.12 ± 0.94
14.04 ± 1.08
5.32-+ 0.70* (P = 0.05)
8.68 ± 1.10" (P = 0.05)
5
9
13.02 ± 2.11
19.84 +- 3.68
6.92-- 0.79* (P = 0.05)
12.24 -+ 2.17" (P = 0.05)
8
12
16.04 ± 1.54
23.68 ± 3.40
8.68 +- 1.08
14.04 ± 2.10
11
*The values of P as indicated for the treated group represent the significance level of the statistical difference between the lipofuscin in the neurons of control and treated mice of the same age after variable periods of treatment. Five mice were sacrificed each time in b o t h treated and control groups. Ten sections were studied in b o t h frontal cortex and h i p p o c a m p u s of each animal.
135
Fig. 4. Nile blue sulfate stain for lipofuscin in the neurons of the cerebral cortex of a 12 month old mouse treated with centrophenoxine for 11 months. (×600)
DISCUSSION One of the most consistent changes in the brain of aging mammals is the deposition of the intracytoplasmic deposition of lipofuscin pigment in the neurons [11, 21-40]. Previous studies in our laboratories have demonstrated a significant reduction of the neuronal lipofuscin pigment by treatment with centrophenoxine for 12 weeks or more in senile guinea pigs [8, 9]. Further studies indicated that the drug also caused a significant improvement in learning and memory in 11-12 months old mice after three months of treatment [10, 17]. This was also associated with a substantial reduction in the lipofuscin in the neurons of cerebral cortex and hippocampus as demonstrated by both light and electron microscopic methods [17]. The effects of the drug on the neuronal lipofuscin has also been observed in the ganglion cells of the nucleus reticularis gigantocellularis by light microscopy [12] and in the neurons and satellite cells of the cervical spinal ganglion cells of the albino rats by electron microscopy [13]. A similar reduction of the pigment was also observed in the neurons of the hypothalamic, hippocampal, reticular nuclei and area postrema of senile guinea pigs [14, 15, 18]. Riga and Riga [16] investigated the effect of the drug on the lipofuscin pigment in the neurons of selected regions from the central nervous system of 24-26 months old rats using fluorescence and histochemical methods. The lipofuscinolytic effects of centrophenoxine by 8 months of treatment in the neurons of different
136 regions of the CNS were 42.3% in pontine reticular formation, 42.0% in cerebral cortex layer V, 27.7% in spinal cord and 25.0% in Purkinje cells in the cerebellum. It appears from the above studies that centrophenoxine is capable of reducing the pigment formation in the neurons of old rodents after several months of treatment. These studies raised several questions. Can the pigment formation be delayed or completely stopped if the treatment is started early in life before its onset? Is the reduction in the lipofuscin pigment in the treated animals due to the slowing of the rate of pigmentogenesis? Can the drug remove the pigments already formed in the neurons? It appears from this study that lipofuscin pigment was always present in the neurons even when the treatment was started before the pigment formation began in young animals. The neurons of both cerebral cortex and hippocampus of the treated mice consistently exhibited less lipofuscin than those in the control animals. The degree of reduction appeared to be dependent on the duration of treatment and a marked reduction was observed after treatment for five months or more (Table I). The lack of significant difference in the pigment levels in earlier periods of treatment might be partly due to small amounts of pigment in the neurons at those ages. Although the drug did not prevent pigment formation in young mice, it appeared to exert its effect by reducing the rate of pigment formation. The question whether the drug is able to remove pigment already formed is not resolved in this study. Since lipofuscin formation in the neurons is a continuous process, the treatment at any age is likely to have some effects on the amount of pigment. However, several other studies have presented evidence in favor of breakdown and dissolution of the pigment particles by the centrophenoxine by electron microscopic methods [10, 14, 15]. Spoerri and Glees [18] demonstrated that pigment particles were transported by cytoplasmic processes and satellite cells, and were finally removed by phagocytes and proliferating endothelial cells of residual capillaries in cultured spinal ganglion neurons and satellite neurons. Similar studies using aging neuroblastoma cells in culture also indicated that the drug reduced the rate of pigment formation and did not stop it even when the treatment was started before the onset of the appearance of the pigment in day 1-3 in culture [19, 20].
ACKNOWLEDGEMENTS The author acknowledges the technical assistance of Miss Nancy Roy. The work was supported by U.S. Public Health Grant NS-11964 and the Veterans Administration research funds. REFERENCES 1 G. ThuiUier, P. Rumpff and J. ThuiUier, Derivatives of acids acting as growth control agents in plants. Pharmacological properties of dimethylaminoethyl ester of acetic acid, C. R. Soc. Biol., 153 (1959) 1914.
2 J. Nickel, U. Breyer and G. Quadbeck, Research on the effects of centophenoxine on the CNS, Int. Congr. Selective Psychostimulant, Rome, 1963. 3 H. Destrem, Clinical trials of centrophenoxine in geriatrics (52 cases), Pr. Med., 69 (1961) 1999.
137 4 J. Delay, J. ThuiUier, P. Pichot, T. Lemperiere and S. Biron, The first results of parachlorophenoxyacetate of dimethylaminoethyl (ANP235) in psychiatry, Ann. Med. Psychol., 118 (1960) 133. 5 G. Nedee, The place of centrophenoxine in human biology, FIR Third Int. Conf., Echos de la Med., Liege, (No. 12) (1961). 6 L. F. Fichez, G. Boureq and G. Nedelec, Therapeutic trial of centrophenoxine in manifestation and accidents of old age, Gaz. Hop., Paris, 133 (1961) 1137. 7 G. Houillon and S. Salles, On the use of centrophenoxine in hospitalized and ambulant patients, Ann. Med-Psychol., 121(1) (1963) 63. 8 K. Nandy and G. H. Bourne, Effect of centrophenoxine on the lipofuscin pigment in the neurons of senile guinea pigs, Nature, 210 (1966) 313-314. 9 K. Nandy, Further study on the effects of centrophenoxine on the lipofuscin pigment in the neurons of senile guinea pigs, J. GerontoL, 23 (1968) 82-92. 10 K. Nandy and H. Lal, Neuronal lipofuscin and learning deficit in aging mammals, in Proc. lOth CoIL Int. Neuropsychopharmacol. Cong., Pergamon Press, New York, 1977. 11 K. Nandy, Properties of lipofuscin pigment in neurons, Acta Neuropathol., 19 (1971) 25-32. 12 K-HV. Chemnitius, G. Machnik, O. Low, M. Arnich and J. Urban, Versuche zur medikamentosen Beeinflussung altersbedingter Veranderungen, Exp. Pathol., 4 (1970) 163-167. 13 C. Meier and P. Glees, Effect of centrophenoxine on the old age pigment in satellite cells and neurons of senile rat spinal ganglia, Acta Neuropathol., 17 (1971) 310-320. 14 M. Hasan, P. Glees and P. E. Spoerri, Dissolution and removal of neuronal lipofusein following dimethylaminoethyl p-chlorophenoxyacetate administration to guinea pigs, Cell Tiss. Res., 150 (1974) 369-375. 15 M. Hasan, P. Glees and E. EI-Ghazzawi, Age-associated changes in the hypothalamus of the guinea pig: Effect of dimethylaminoethyl p-chlorophenoxyacetate. An electron microscopic and histochemical study, Exp. Gerontol., 9 (1974) 153-159. 16 S. Riga and D. Riga, Effects of centrophenoxine on the lipofuscin pigments in the nervous system of old rats, Brain Res., 72 (1974) 265-275. 17 K. Nandy, Effects of centrophenoxine in aging mammalian brain, Z Am. Gerontol. Soc., 26 (1978) 74-81. 18 P. E. Spoerri and P. Glees, The effects of dimethylaminoethyl p-chlorophcnoxyacetate on spinal ganglia neurons and satellite cells in culture. Mitochondrial changes in the aging neurons. An electron microscopic study, Mech. Ageing Dev., 3 (1974) 131-155. 19 K. Nandy and H. Schneider, Lipofuscin pigment formation in neuroblastoma cells in culture, in R. D. Terry and S. Gershon (eds.), Neurobiology of Aging, Raven Press, New York, 1976, pp. 245-264. 20 H. Schneider and K. Nandy, Effects of centrophenoxine on lipofuscin formation in neuroblastoma cells in culture, Z Gerontol., 32 (1977) 132-139. 21 T. Samorajski, J. R. Keefe and J. M. Ordy, Intracellular localization of lipofuscin age pigments in the nervous system, J. Gerontol., 19 (1964) 262-276. 22 T. Samorajski, J. M. Ordy and J. R. Keefe, The fine structure of lipofuscin age pigment in the nervous system of aged mice, J. Cell Biol., 26 (1965) 779-795. 23 H. Brody, The deposition of aging pigment in the human cerebral cortex, J. GerontoL, 16 (1960) 258-261. 24 B. S. Nanda and R. Getty, Lipofuscin pigment in the nervous system of aging pig, Aging Pigment, Curt. Res., I (1974) 11-18. 25 W. B. Weglicki, W. Reichel and P. P. Nair, Accumulation of lipofuscinqike pigment in the rat adrenal gland as a function of vitamin E deficiency, Z Gerontol., 21 (1966) 469-475. 26 R. Whiteford and R. Getty, Distribution of lipofuscin in the canine and porcine brain as related to aging, J. Gerontol., 21 (1966) 31-44. 27 K. R. Brizzee, J. M. Ordy, J. Hansche and B. Kaack, Quantitative assessment of changes in neurons and gliai cells packing density and lipofuscin accumulation with age in the cerebral cortex of a nonhuman primate (Macaca mulatta), in R. Terry and S. Gershon (eds.), Neurobiology o f Aging, Raven Press, New York, 1976, pp. 229-244. 28 S. S. Sekhon and D. S. Maxwell, Ultrastructural changes in neurons of the spinal anterior horn of aging mice with particular references to the accumulation of lipofuscin pigment, J. Neurocytol., 3 (1974) 59-72.
138 29 H. Lal, S. Pogacar, P. R. Daly and S. Puri, Behavioral and neuropathological manifestations of nutritionally induced central nervous system aging in the rat, in D. Ford (ed.), Neurobiological Aspects of Maturation andAging, Elsevier, New York, 1973, pp. 129-140. 30 H. Hamperl, Histochemical studies on the lipofuscin pigments in human heart, Arch. Pathol. Anat. PhysioL, 327 (1934) 112. 31 N. M. Sulkin, The occurrence and duration of senile pigment experimentally induced in the nerve cells of the young rat, Anat. Rec., 130 (1958) 377. 32 K. W. O'Steen and K. Nandy, Lipofuscin pigment in neurons of young mice with a hereditary central nervous system defect, Am. J. Anat., 128 (1970) 359-366. 33 A. N. Siakotos, I. Watanaba, A. Siato and S. Fleischer, Procedure of the isolation of two distinct lipopigments from human brain: lipofuscin and ceroid, Biochem. Meal., 4 (1970) 361-375. 34 W. Reichel and R. Garcia-Bunel, Pathologic findings in progeria: Myocardial fibrosis and lipofuscin pigment, Am. J. Clin. Pathol., 53 (1970) 243. 35 W. Reichel, J. Hollander, J. H. Clark and B. L. Strehler, Lipofuscin pigment accumulation as function of age and distribution in rodent brain, J. Gerontol., 23 (1968) 71-78. 36 M. Hasan and P. Glees, Ultrastructural age changes in hippocampal neurons, synapses and neuroglia, Exp. GerontoL, 8 (1973) 75-83. 37 M. Hasan and P. Glees, Electron microscopical appearance of neuronal lipofuscin using different preparation techniques including freeze-etching, Exp. Gerontol., 7 (1972) 345-351. 38 W. Reichel, J. Hollander, J. H. Clark and B. L. Strehler, Lipofuscin pigment accumulation as a function of age and distribution in rodent brain, J. GerontoL, 23 (1968) 71-78. 39 G. Gopinath and P. Glees, Mitochondrial genesis of lipofuscin in the mesencephalic nucleus of the V nerve of the aged rats, ActaAnat., 89 (1974) 14-20. 40 D. M. A. Mann and P. O. Yates, Lipofuscin pigments - their relationship to ageing in the human nervous system. I. The lipofuscin content of nerve cells, Brain, 97 (1974) 481-488.
131
©Elsevier Sequoia S.A., Lausanne - Printed in the Netherlands
L I P O F U S C I N O G E N E S I S IN M I C E E A R L Y T R E A T E D W I T H C E N T R O PHENOXINE
K. NANDY Geriatric Research, Education and Clinical Center, Veterans Administration Hospital, Bedford, Mass. (U.S.A.)
(Received August 26, 1977;in revised form January 9, 1978)
SUMMARY Previous studies in our and other laboratories indicated that there is a reduction in the neuronal lipofuscin in old rodents after several weeks of treatment with centrophenoxine. The present study investigates whether this chemical can prevent pigment formation if given early in life before the onset of pigmentogenesis. The study shows that the drug did not stop lipofuscin formation in 1 month old mice. But there was a consistent decrease in the pigment in the neurons of cerebral cortex and hippocampus of the treated animals compared to the age-matched controls. The degree of reduction was largely dependent on the duration of the treatment and a significant diminution was noted after treatment for five months or more.
INTRODUCTION Centrophenoxine is synthesized as the hydrochloride of the dimethylaminoethyl ester of p-chlorophenoxyacetic acid. The former component of the drug is a synthetic substance chemically related to the plant growth hormone auxin and the latter is a natural substance in our body and is a precursor of acetylcholine. Clinical trials on geriatric patients in Europe demonstrated a marked improvement of symptoms of senility such as confusion, psychosomatic asthenia and disturbances of memory after treatment for several months with a dose of 80 mg/kg of body weight [1-7]. Its effect on the lipofuscin pigment in the neurons has been demonstrated in the aged guinea pig and mice and a marked reduction of the pigment was noted after treatment for 12 weeks or more [8-18]. In this paper, an attempt has been made to determine whether centrophenoxine can prevent lipofuscin pigment formation completely if the treatment is started in young mice prior to the onset of pigmentogenesis.
132 MATERIALSAND METHODS About 100 old female C57BL/6 mice (1 month) were used in this study. Animals were maintained in constant temperature and humidity controlled environmental chamber and were fed on Purina mouse chow. Fifty female mice (1 month) were treated with centrophenoxine by intraperitoneal injection (80 mg/kg of body weight) daily for 2-11 months and a similar number of the same age and sex and injected intraperitoneally with normal saline were used as controls. Five mice of the control groups were sacrificed at the beginning of the experiment and five mice belonging to each of the control and treated groups were sacrificed after 2, 5, 8, and 11 months of treatment. After the animals were sacrificed, their brains were dissected out and fixed in 10% neutral buffered formalin. Frozen 10/am sections were cut in a cryostat and lipofuscin pigment was visualized by its characteristic autofluorescence by exposing the section to u.v. light at a wavelength of 365 nm. The periodic acid-Schiff and Nile blue sulfate methods also demonstrated the pigment. Semi-quantitative measurements of the pigments in sections were carried out by counting the number of intersections overlying the pigment particles in each of ten sections of frontal cerebral cortex and hippocampus using an ocular grid in a comparison microscope (Duostar, AO).
Fig. 1. Nile blue sulfate stain for lipofuscin in the neurons of the cerebral cortex of a 6 month old control mouse. (x600)
133 RESULTS There were hardly any lipofuscin pigment in the neurons of the cerebral cortex and hippocampus in 1 month old mice. The pigment formation began to appear at 2-3 months of age and thereafter increased progressively as a function of age. The pigment was diffusely distributed in the cytoplasm of the neurons at 2-5 months of age and tended to form perinuclear or polar clumps as the animals grew older. The pigment was primarily visualized by its characteristic autofluorescence and also by histochemical staining with PAS and Nile blue sulfate. In general, lipofuscin was consistently observed in larger quantities in the neurons of the hippocampus than in the cerebral cortex. The treatment with centrophenoxine was started at the age of 1 month when the neurons of cerebral cortex and hippocampus demonstrated very little lipofuscin. Five mice were sacrificed after 2, 5, 8, and 11 months o f treatment and lipofuscin pigment was measured by the semi-quantitative method using an ocular grid in a microscope. The amount of pigment was uniformally lower in all treated groups compared to the controls o f the same age by all the methods employed (Figs. 1--4). While lipofuscin was demonstrated in only 3 out of 5 mice in'the treated group at 3 months of age, all control animals exhibited the pigment in the neurons. It was specially noted that the difference in the pigment content in the treated and control mice was most marked after 11 months of treatment and was statistically significant after treatment for 5 months or more (Table I).
Fig. 2. Nile blue sulfate stain for lipofuscin in the neurons of the cerebral cortex of a 6 month old mouse treated with centrophenoxine for 5 months. (X 600)
134
Fig. 3. Nile blue sulfate stain for lipofuscin in the neurons of the cerebral cortex of a 12 m o n t h old control mouse. (× 600)
TABLE I LIPOFUSCIN PIGMENT COUNTED BY OCULAR GRID METHOD IN THE NEURONS OF C E R E B R A L CORTEX AND HIPPOCAMPUS OF MICE T R E A T E D WITH CENTROPHENOXINE AND AGE-MATCHED CONTROLS
Control group Age (months)
Treated group Neurons of frontal cortex +- 0.34
Neurons of hippocampus
0.6
+- 0.66
Neurons of frontal cortex
Neurons of hippocampus
Duration of treatment (months)
-
-
-
1
0.4
3
4.80 -+ 0.34
6.96 +- 1.08
2.96 -+ 0.98* (P = 0.4)
4.76 ± 1.60" (P = 0.2)
2
6
10.12 ± 0.94
14.04 ± 1.08
5.32-+ 0.70* (P = 0.05)
8.68 ± 1.10" (P = 0.05)
5
9
13.02 ± 2.11
19.84 +- 3.68
6.92-- 0.79* (P = 0.05)
12.24 -+ 2.17" (P = 0.05)
8
12
16.04 ± 1.54
23.68 ± 3.40
8.68 +- 1.08
14.04 ± 2.10
11
*The values of P as indicated for the treated group represent the significance level of the statistical difference between the lipofuscin in the neurons of control and treated mice of the same age after variable periods of treatment. Five mice were sacrificed each time in b o t h treated and control groups. Ten sections were studied in b o t h frontal cortex and h i p p o c a m p u s of each animal.
135
Fig. 4. Nile blue sulfate stain for lipofuscin in the neurons of the cerebral cortex of a 12 month old mouse treated with centrophenoxine for 11 months. (×600)
DISCUSSION One of the most consistent changes in the brain of aging mammals is the deposition of the intracytoplasmic deposition of lipofuscin pigment in the neurons [11, 21-40]. Previous studies in our laboratories have demonstrated a significant reduction of the neuronal lipofuscin pigment by treatment with centrophenoxine for 12 weeks or more in senile guinea pigs [8, 9]. Further studies indicated that the drug also caused a significant improvement in learning and memory in 11-12 months old mice after three months of treatment [10, 17]. This was also associated with a substantial reduction in the lipofuscin in the neurons of cerebral cortex and hippocampus as demonstrated by both light and electron microscopic methods [17]. The effects of the drug on the neuronal lipofuscin has also been observed in the ganglion cells of the nucleus reticularis gigantocellularis by light microscopy [12] and in the neurons and satellite cells of the cervical spinal ganglion cells of the albino rats by electron microscopy [13]. A similar reduction of the pigment was also observed in the neurons of the hypothalamic, hippocampal, reticular nuclei and area postrema of senile guinea pigs [14, 15, 18]. Riga and Riga [16] investigated the effect of the drug on the lipofuscin pigment in the neurons of selected regions from the central nervous system of 24-26 months old rats using fluorescence and histochemical methods. The lipofuscinolytic effects of centrophenoxine by 8 months of treatment in the neurons of different
136 regions of the CNS were 42.3% in pontine reticular formation, 42.0% in cerebral cortex layer V, 27.7% in spinal cord and 25.0% in Purkinje cells in the cerebellum. It appears from the above studies that centrophenoxine is capable of reducing the pigment formation in the neurons of old rodents after several months of treatment. These studies raised several questions. Can the pigment formation be delayed or completely stopped if the treatment is started early in life before its onset? Is the reduction in the lipofuscin pigment in the treated animals due to the slowing of the rate of pigmentogenesis? Can the drug remove the pigments already formed in the neurons? It appears from this study that lipofuscin pigment was always present in the neurons even when the treatment was started before the pigment formation began in young animals. The neurons of both cerebral cortex and hippocampus of the treated mice consistently exhibited less lipofuscin than those in the control animals. The degree of reduction appeared to be dependent on the duration of treatment and a marked reduction was observed after treatment for five months or more (Table I). The lack of significant difference in the pigment levels in earlier periods of treatment might be partly due to small amounts of pigment in the neurons at those ages. Although the drug did not prevent pigment formation in young mice, it appeared to exert its effect by reducing the rate of pigment formation. The question whether the drug is able to remove pigment already formed is not resolved in this study. Since lipofuscin formation in the neurons is a continuous process, the treatment at any age is likely to have some effects on the amount of pigment. However, several other studies have presented evidence in favor of breakdown and dissolution of the pigment particles by the centrophenoxine by electron microscopic methods [10, 14, 15]. Spoerri and Glees [18] demonstrated that pigment particles were transported by cytoplasmic processes and satellite cells, and were finally removed by phagocytes and proliferating endothelial cells of residual capillaries in cultured spinal ganglion neurons and satellite neurons. Similar studies using aging neuroblastoma cells in culture also indicated that the drug reduced the rate of pigment formation and did not stop it even when the treatment was started before the onset of the appearance of the pigment in day 1-3 in culture [19, 20].
ACKNOWLEDGEMENTS The author acknowledges the technical assistance of Miss Nancy Roy. The work was supported by U.S. Public Health Grant NS-11964 and the Veterans Administration research funds. REFERENCES 1 G. ThuiUier, P. Rumpff and J. ThuiUier, Derivatives of acids acting as growth control agents in plants. Pharmacological properties of dimethylaminoethyl ester of acetic acid, C. R. Soc. Biol., 153 (1959) 1914.
2 J. Nickel, U. Breyer and G. Quadbeck, Research on the effects of centophenoxine on the CNS, Int. Congr. Selective Psychostimulant, Rome, 1963. 3 H. Destrem, Clinical trials of centrophenoxine in geriatrics (52 cases), Pr. Med., 69 (1961) 1999.
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